In 1987, matrix-assisted laser desorption/ionization mass spectrometry (MALDI) was introduced by Hillenkamp and Karas, and since has become a very powerful bioanalytical tool (Anal. Chem. 60:2288-2301, 1988; see also Burlingame et al., Anal. Chem. 68:599-651, 1996 and references cited therein). The success of MALDI in the area of protein science can be attributed to several factors. The greatest of these is that MALDI can be rapidly (.about.5 minutes) applied as an analytical technique to analyze small quantities of virtually any protein (practical sensitivities of .about.1 pmole protein loaded into the mass spectrometer). The technique is also extremely accurate. Beavis and Chait demonstrated that the molecular weights of peptides and proteins can be determined to within .about.0.01% by using methods in which internal mass calibrants (x-axis calibration) are introduced into the analysis (Anal Chem. 62:1836-40, 1990). MALDI can also be made quantitative using a similar method in which internal reference standards are introduced into the analysis for ion signal normalization (y-axis calibration). Quantitative determination of proteins and peptides is possible using this approach with accuracy's on the order of .about.10% (Nelson et al., Anal. Chem. 66:1408-15, 1994). Finally, MALDI is extremely tolerant of large molar excesses of buffer salts and, more importantly, the presence of other proteins.
With the high tolerance towards buffer salts and other biomolecular components comes the ability to directly analyze complex biological mixtures. Many examples exist where LDI is used to directly analyze the results of proteolytic or chemical digestion of polypeptides (see Burlingame et al., supra). Other examples extend to elucidating post-translational modifications (namely carbohydrate type and content), a process requiring the simultaneous analysis of components present in a heterogeneous glycoprotein mixture (Sutton et al., Techniques in Protein Chemisty III, Angeletti, Ed., Academic Press, Inc., New York, pp. 109-116, 1993). Arguably, the most impressive use of direct mixture analysis is the screening of natural biological fluids. In that application, proteins are identified, as prepared directly from the host fluid, by detection at precise and characteristic mass-to-charge (m/z) values (Tempst et al., Mass Spectrometry in the Biological Sciences, Burlingame and Carr, Ed., Humana Press, Totowa, N.J., p. 105, 1996).
While the above examples involving direct MALDI analysis of complex mixtures are, in their own right, quite impressive, there exist limits to the extent of practical application. These limits are reached when a target analyte is a minor component of the mixture, and is present at low concentration. A common occurrence in such situations is that the target analyte is never observed in the MALDI mass spectrum. This lack of detection is generally due to the low concentration of the analyte yielding ion signals at or below the instrumental limits of detection--an effect further exacerbated by protein-analyte interactions "stealing" analyte molecules from the MALDI process, and/or high instrumental baselines produced from other proteins present in the mixture ("analyte masking"). Methods for the selective concentration of specific species in the mixture (prior to MALDI) are therefore required in order to achieve ion signals from the target analyte.
The use of an affinity ligand-derivatized support to selectively retrieve a target analyte specifically for MALDI analysis was first demonstrated by Hutchens and Yip (Rapid Commun. Mass Spectrom. 7:576-80, 1993). Those investigators used single-stranded DNA-derivatized agarose beads to selectively retrieve a protein, lactoferrin, from pre-term infant urine by incubating the beads with urine. The agarose beads were then treated as the MALDI analyte--a process involving mixing with a solution-phase MALDI matrix followed by deposition of the mixture on a mass spectrometer probe. MALDI then proceeded in the usual manner. Results indicated that the derivatized beads selectively retrieved and concentrated the lactoferrin; enough so to enable ion signal in the MALDI mass spectrum adequate to unambiguously identify the analyte at the appropriate m/z value (81,000 Da). A number of variations on this approach have since been reported. These include the use of immunoaffinity precipitation for the MALDI analysis of transferring in serum Nakanishi et al., Biol. Mass Spectrom. 23:230-33, 1994), screening of ascites for the production of monoclonal antibodies (Papac et al., Anal. Chem. 66:2609-13, 1994), and the identification of linear epitope regions within an antigen (Zhao et al., Anal. Chem. 66:3723-26, 1994). Even more recently, the affinity capture approaches have been made rigorously quantitative by incorporating mass-shifted variants of the analyte into the analysis (Nelson et al. Anal. Chem. 67:1153-58, 1995). The variants are retained throughout the analysis (in the same manner as the true analyte) and observed as unique (resolved) signals in the MALDI mass spectrum. Quantitation of the analyte is performed by equating the relative ion signals of the analyte and variant to an analyte concentration.
The affinity capture techniques discussed above make use of "off-line" incubation steps. That is, the target analyte is captured on some form of affinity-derivatized support (generally chromatographic beads) and then eluted onto a mass spectrometer probe tip. Use of such off-line approaches have generally been considered advantageous because relatively large quantities of affinity-derivatized reagent (and hence large quantities of affinity ligand) can be used in a given analysis. These techniques can also be performed in a flowing manner, with large volumes of analyte solution and wash buffers brought in contact with the affinity reagent. As a result, the affinity capture steps are fast (.about.5 min) and clean (low non-specific binding).
An attractive alternative to using beaded material in an off-line approach is to incorporate the affinity ligand directly onto the mass spectrometer probe element. This would allow the probe to be used to selectively retrieve and retain an analyte from solution, and then be MALDI-analyzed (bypassing any elution steps). Although there have been some attempts to use a LDI mass spectrometer probe element as the affinity-capture device (Brockman et al., Anal. Chem. 67:4581-85, 1995), such attempts have fallen short of expectation. This downfall is due mainly to the severe limitation on the number of surface-active affinity sites possible on the essentially two-dimensional surface of the mass spectrometer probe. As a result, such derivatized-probes are neither as sensitive or as rapid as the off-line approaches. A second, more fundamental issue deals with analyte utilization. In the aforementioned analyses, affinity interactions are used solely to define the species introduced into the mass spectrometer--mass spectrometry, as a destructive technique, then destroys the analyte.
Accordingly, there still exists a need in the art for improved analytical techniques, particularly in the field of mass spectrometry. Such techniques should be capable of analyzing complex mixtures while overcoming the disadvantages associated with existing off-line incubation steps, as well as provide information in addition to mere species identification. The present invention fulfills these needs, and provides further related advantages.